Increasing the MHC I affinity of SSX2-p103 resulted in increased activation, expansion, and PD-1 expression

A) T2 cells were incubated with the indicated peptides for six hours, stained for HLA-A2, and analyzed by flow cytometry. HLA-A2 MFI is displayed as the fold change over cells that were incubated with a non-specific peptide B) Mice were immunized with each peptide and after one week the spleens were collected. The percentage of CD8 T cells that were positive for SSX2-p103-tetramer staining was determined. C) Splenocytes from immunized mice were stimulated with the native SSX2-p103 peptide, and 4-1BB expression and intracellular cytokine staining for one or more Th1 (IFNγ, TNFα, IL2) cytokines, was assessed by flow cytometry. D) PD-1 amounts on the tetramer+ cells from B. APL abbreviations are shown in . Letters along the X axis indicate AA changes in the SSX2-p103 sequence as indicated in . Nat = SSX2-p103 native sequence, Flu = HLA-A2 restricted flu epitope positive control. Results shown are representative of two independent experiments with N=6 replicates each, *= P < 0.05, two-way ANOVA with Bonferroni’s posttest. Representative flow cytometry gating strategy shown in .

A) TAP-deficient RMA-S cells were incubated with the indicated peptides () for six hours, at which time the cells were stained for MHC-I and analyzed by flow cytometry. MHC-I MFI is displayed as the fold change over cells that were incubated with a non-specific peptide. B) OT-1 mouse splenocytes were incubated with the indicated peptides and cytokine expression was assessed by intracellular cytokine staining. The number of cells expressing IL2 and/or TNFα and/or IFNγ is shown as the percent of total CD8 T cells. Highlighted peptides were selected for further analysis. The letters along the x axis indicate AA changes in the SIINFEKL sequence as indicated in , ex. IP-FT = SIPNTEKL. No = vehicle control, NS = non-specific peptide control, SSX2 p103–111 (RLQGISPKI), PMA = PMA and ionomycin positive control. Results shown are representative of two independent experiments with N=6 replicates each, *= P < 0.05, two-way ANOVA followed by Bonferroni’s posttest correction. Representative flow cytometry gating strategy shown in .

Vaccination with high-affinity peptides led to persistent PD-1 and LAG3 expression in vivo

A) C57BL/6 mice received 2×106 OT-1 cells by adoptive transfer and the next day were immunized with 100µg OVA, 100µg of one of the variants, or 0.1 µg (low dose) of OVA with Freund’s adjuvant (top, CFA) or alone in PBS (bottom, vehicle). Spleens were harvested from individual groups every two days for six days. The MFI of indicated receptors was assessed on SIINFEKL-tetramer+ CD45+ CD8+ T cells directly ex vivo. B) C57BL/6 mice were immunized as in panel A with 100µg FT, OVA, or NS control in CFA. Expression of 4-1BB (activation), PD-1, and LAG3 were assessed on splenocytes on days 2, 7, 14 and 28. Results shown are representative of two independent experiments with N=5 mice/group. Statistical comparisons are made between groups receiving OVA and FT. *= P < 0.05, two-way ANOVA with Bonferroni correction. (NS = non-specific peptide, SSX2 p103–111 (RLQGISPKI)). Representative flow cytometry gating strategy shown in .

Top) APCs were stained with CellTracker red (CMTPX), incubated with the indicated peptides or various concentrations of OVA (ng/mL), and allowed to incubate for one hour. Isolated OT-1 T cells stained with CellTracker green (CMFDA) were added to the peptide loaded APCs. Upon addition of the T cells, 20× time-lapse microscopy was conducted for one hour at one minute intervals to monitor APC:T-cell contact times. Images are representative of cells interacting for the mean time observed with the respective peptide. Color was removed from the surrounding cells for ease of tracking. Bottom) Contact time for each peptide was quantified. Prior to imaging, cells were removed, allowed to incubate together for 72 hours and analyzed by flow cytometry. Association between contact time and peptide affinity was determined via two-sided Kendall’s rank correlation. NS = non-specific peptide, SSX2 p103–111 (RLQGISPKI). * = P < 0.05, two-way ANOVA followed by Bonferroni correction. For contact time calculations, N=30 APC:T-cell interactions were evaluated for each experimental condition. For MFI of 4-1BB, PD-1 and LAG3 expression in the bottom panels, data shown is for 6-well replicates. Representative flow cytometry gating strategy shown in .